US9499591B2 - Truncated L1 protein of human papillomavirus type 52 - Google Patents

Truncated L1 protein of human papillomavirus type 52 Download PDF

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US9499591B2
US9499591B2 US13/807,858 US201113807858A US9499591B2 US 9499591 B2 US9499591 B2 US 9499591B2 US 201113807858 A US201113807858 A US 201113807858A US 9499591 B2 US9499591 B2 US 9499591B2
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protein
hpv52
truncated
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solution
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Shaowei Li
Xiaobing Mo
Minxi Wei
Huirong Pan
Jun Zhang
Ningshao Xia
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Xiamen University
Xiamen Innovax Biotech Co Ltd
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Xiamen Innovax Biotech Co Ltd
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    • C07K14/01—DNA viruses
    • C07K14/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • A61K2039/525—Virus
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2710/20051—Methods of production or purification of viral material

Definitions

  • the invention relates to the field of molecular virology and immunology.
  • the invention relates to a truncated L1 protein of Human Papillomavirus Type 52, its coding sequence and preparation method, and a virus-like particle comprising the protein, wherein the protein and the virus-like particle are useful for preventing HPV (particularly HPV52) infection, and a disease caused by HPV (particularly HPV52) infection, such as cervical cancer.
  • the invention also relates to the use of the protein and the virus-like particle in the preparation of a pharmaceutical composition or a vaccine for preventing HPV (particularly HPV52) infection, and a disease caused by HPV (particularly HPV52) infection, such as cervical cancer.
  • HPV Human Papillomavirus
  • DNA deoxyribonucleic acid
  • ORFs open reading frames
  • the genome can be divided into three parts in terms of function: (1) the early region (E), approximately 4.5 Kb in length, coding for 6 non-structural proteins E1, E2, E4-E7 associated with virus replication, transcription and transformation; (2) the late region (L), approximately 2.5 Kb in length, coding for the major capsid protein L1 and the minor capsid protein L2; (3) the long control region (LCR), located between the end of the L region and the initiating terminal of the E region, approximately 800-900 bp in length, and comprising regulator elements for DNA replication and expression instead of coding for proteins.
  • HPV viral particles have a diameter of 45-55 nm, wherein the nucleocapsid, consisting of L1 and L2, exhibits icosahedral symmetry and comprises 72 capsomers.
  • HPV types are divided into three groups depending on their relation with tumorigenesis: (1) group of low or no cancerogenic risk, containing HPV 6, 11, 39, 41, 42, and 43; (2) group of medium cancerogenic risk, containing HPV 31, 33, 35 and 51; and (3) group of high cancerogenic risk, containing HPV 16, 18, 58, 45 and 52.
  • HPV molecular epidemiological investigation demonstrates that infection by high-risk HPV types is an important factor responsible for the development of cervical cancer.
  • HPV DNA is detected in over 80% of them.
  • Cervical cancer is a common malignant tumor among women, the incidence of which is only next to breast cancer, and seriously threatens the health of women.
  • Cases in developing countries account for approximately 83% of the total cervical cancer cases. In these developing countries, the cervical cancer cases account for about 15% of female malignant tumors, in contrast to 1.5% in developed countries.
  • Cervical cancer is most prevalent in sub-Saharan Africa, central and Southern Asia, Latin America, and Eastern Asia. Cervical cancer is also prevalent in China. The incidence of cervical cancer among married women is as high as 1026/100000 in Lueyang County of Shanxi City.
  • HPV 16 and 18 subtypes are the most common types in cervical cancer worldwide, and HPV52 subtype is the sixth most common high-risk HPV type.
  • HPV52 is a high-risk cancerogenic HPV type only next to HPV 16, 33 and 18.
  • HPV vaccines are Gardasil® from Merck and Cervarix® from GSK, which comprise HPV6/11/16/18 and HPV16/18 VLP, respectively, but do not comprise HPV type 52 VLP.
  • vaccines directed to HPV type 52 shall be involved in the development of vaccines for high-risk types, which cover a wider scope and are more suitable for Chinese population.
  • HPV L1 protein with a molecular weight of 55-60 kDa, is the major capsid protein of the human papillomavirus and the main target protein of the HPV vaccine.
  • HPV L1 protein expressed in many expression systems can form Virus-Like Particles (VLPs) which resemble native HPV particles morphologically, without the assistance of the L2 protein.
  • the VLPs consisting of 72 pentamers of the L1 proteins, exhibit icosahedral symmetry. Since the VLPs retain the native epitopes of the viral particles, they are highly immunogenic and can induce the generation of neutralization antibodies against homologous HPV (Kirnbauer, R., F. Booy, et al. 1992 Proc Natl Acad Sci USA 89(24): 12180-4). Furthermore, the VLPs are safe and have no potential cancergenic risk as they contain no viral nucleic acids. Therefore, VLP vaccines have become the primary candidate for HPV vaccines.
  • HPV L1 protein expressed in eukaryotic expression systems shows little conformational difference from that of the native virus, and can self-assemble into VLPs.
  • purified VLPs can be easily obtained after simple gradient density centrifugation. It brings a lot of convenience to the purification work.
  • HPV vaccine Gardasil® which came into the market recently, is more expensive than others due to low expression level and high production cost of the Saccharomyces cerevisiae expression system employed in its manufacture, and therefore, its general application is limited.
  • HPV L1 protein in a prokaryotic expression system such as E. coli expression system has been previously reported.
  • HPV VLPs can be obtained from the proteins by steps such as purification from inclusion bodies and renaturation (Kelsall, S. R. and J. K. Kulski (1995).
  • HPV L1 protein may be expressed in a soluble form with a correct conformation in E. coli and be dissolved in the supernatants of E. coli lysate, the expression level is low. Moreover, since there are large number and amounts of impure proteins, it is difficult to isolate the proteins of interest from them. Although it is also reported that the expression level of L1 protein can be increased in the supernatants by means of GST fusion expression and the purification of the protein of interest is facilitated (Li, M., T. P. Cripe, et al. (1997), J Virol 71(4): 2988-95), it still cannot be applied to larger-scale production because expensive enzymes are required to cleave the fusion protein.
  • a truncated HPV52 L1 protein capable of inducing the generation of neutralization antibodies against HPV52 can be expressed in an E. coli expression system on a large scale, wherein the truncated HPV52 L1 protein can be produced with a high yield, and the purity of the purified protein reaches at least 50% or higher (such as 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99%). Moreover, further treatment of the purified protein results in the obtainment of VLPs capable of inducing the generation of protective antibodies against HPV52.
  • the invention relates to a truncated HPV52 L1 protein or variants thereof, wherein said protein has 27-42 amino acids, for example, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids, truncated at its N-terminal.
  • the invention relates to a truncated HPV52 L1 protein or variants thereof, wherein said protein has 27-42 amino acids, for example, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids, truncated at its N-terminal, as compared with wild type HPV52 L1 protein.
  • the truncated HPV52 L1 protein has 27-42 amino acids (such as, 35-42 amino acids), for example, 27, 35, 38, 40, or 42 amino acids, truncated at its N-terminal, as compared with wild type HPV52 L1 protein.
  • the truncated HPV52 L1 protein has 40 amino acids truncated at its N-terminal, as compared with wild type HPV52 L1 protein.
  • the truncated HPV52 L1 protein (cited hereafter as the truncated protein) has an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13; such as, an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 13.
  • the truncated protein has an amino acid sequence as set forth in SEQ ID NO: 12.
  • the invention relates to a polynucleotide encoding the truncated protein or variants thereof according to the invention, and a vector containing the polynucleotide.
  • Vectors for inserting a polynucleotide of interest are well known in the art, including, but not limited to clone vectors and expression vectors.
  • the vectors are, for example, plasmids, phages, cosmids, etc.
  • the invention also relates to a host cell comprising the polynucleotide or vector as described above.
  • the host cell includes, but is not limited to prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, for example, mouse cells, human cells, etc.).
  • the host cell according to the invention may also be a cell line, such as 293T cell.
  • the invention relates to a HPV52 virus-like particle, comprising or consisting of or formed from the truncated protein or variants thereof according to the invention.
  • the HPV52 virus-like particle according to invention comprises or is consisted of or formed from the truncated HPV52 L1 protein having 27-42 amino acids, for example, 27, 35, 38, 40, or 42 amino acids, truncated at its N-terminal, as compared with wild type HPV52 L1 protein.
  • the HPV52 virus-like particle according to invention comprises or is consisted of or formed from the truncated HPV52 L1 protein having a sequence as set forth in SEQ ID NO: 1, 7, 10, 12, or 13.
  • the invention also relates to a composition
  • a composition comprising said truncated protein or variants thereof, or said polynucleotide or vector or host cell or HPV52 virus-like particle.
  • the composition comprises the truncated protein or variants thereof according to the invention.
  • the composition comprises the HPV52 virus-like particle according to the invention.
  • the invention also relates to a pharmaceutical composition or vaccine comprising the HPV52 virus-like particle according to invention, and optionally pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition or vaccine according to the invention is useful for preventing HPV (particularly HPV52) infection, and a disease caused by HPV (particularly HPV52) infection, such as cervical cancer.
  • the HPV52 virus-like particle is present at an amount effective for preventing HPV infection or cervical cancer.
  • the pharmaceutical composition or vaccine according to the invention further comprises at least one virus-like particle selected from the group consisting of HPV6 L1 protein virus-like particle, HPV11 L1 protein virus-like particle, HPV16 L1 protein virus-like particle, HPV18 L1 protein virus-like particle, HPV31 L1 protein virus-like particle, HPV33 L1 protein virus-like particle, HPV45 L1 protein virus-like particle, and HPV58 L1 protein virus-like particle; preferably these virus-like particles are independently present at an amount effective for preventing cervical cancer or infection by the corresponding HPV subtype.
  • the pharmaceutical composition or vaccine according to the invention may be administrated by methods well known in the art, for example, but not limited to, orally or by injection.
  • the particularly preferred administration route is injection.
  • each unit dosage contains 5 ⁇ g-80 ⁇ g, preferably 20 ⁇ g-40 ⁇ g HPV52 virus-like particle.
  • the invention relates to a method for obtaining the truncated protein according to the invention, comprising expressing the truncated protein according to the invention with an E. coli expression system, and carrying out a purification process on the lysis supernatant containing the truncated protein,
  • the method for obtaining the truncated protein according to the invention comprises
  • the salt concentration in b) is from 200 mM to 500 mM.
  • the invention also relates to a method for obtaining the HPV52 virus-like particle according to invention, on the basis of the obtainment of the truncated protein of the invention, comprising the steps of:
  • the invention also relates to a method for preparing a vaccine, comprising blending the HPV52 virus-like particle according to the invention, and optionally, one or more virus-like particles selected from the group consisting of virus-like particles of HPV types 6, 11, 16, 18, 31, 33, 45 and 58, with pharmaceutically acceptable carriers and/or excipients.
  • the vaccine obtained is useful for preventing HPV (particularly HPV52) infection, and a disease caused by HPV (particularly HPV52) infection, such as cervical cancer.
  • the invention in another aspect, relates to a method for preventing HPV infection or a disease caused by HPV infection, comprising administrating a prophylactically effective amount of the HPV52 virus-like particle or pharmaceutical composition or vaccine according to the invention.
  • the HPV infection is HPV52 infection.
  • the disease caused by HPV infection includes, but is not limited to cervical cancer.
  • the subject is mammalian, such as human.
  • the invention also relates to the use of the truncated protein or variants thereof or the HPV52 virus-like particle according to invention in the preparation of a pharmaceutical composition or vaccine for preventing HPV infection or a disease caused by HPV infection.
  • the HPV infection is HPV52 infection.
  • the disease caused by HPV infection includes, but is not limited to cervical cancer.
  • the invention also relates to the truncated protein or variants thereof or the HPV52 virus-like particle according to invention, for use in the prevention of HPV infection or a disease caused by HPV infection.
  • the HPV infection is HPV52 infection.
  • the disease caused by HPV infection includes, but is not limited to cervical cancer.
  • a protein having X amino acids truncated at its N-terminal refers to a protein resulted from substituting the amino acid residues from positions 1 to X at the N-terminal of the protein with methionine residue encoded by an initiator codon (for initiating protein translation).
  • a HPV52 L1 protein having 27 amino acids truncated at its N-terminal refers to a protein resulted from substituting the amino acid residues from positions 1 to 27 at the N-terminal of wild type HPV52 L1 protein with methionine residue encoded by an initiator codon.
  • the term “variant” refers to a protein, whose amino acid sequence is different from the truncated HPV52 L1 protein according to the invention (for example, the protein as set forth in SEQ ID NO: 1, 7, 10, 12, or 13) by one or more (for example, 1-10, or 1-5 or 1-3) amino acids (such as conservative amino acid substitutions), or which has an identity of at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the truncated HPV52 L1 protein according to the invention (for example, the protein as set forth in SEQ ID NO: 1, 7, 10, 12, or 13), and which retains the essential characteristics of the truncated protein.
  • essential characteristics may be one or more of the following characteristics: capable of inducing the generation of neutralization antibodies against HPV52; capable of being expressed in E. coli in a soluble manner; capable of obtaining purified protein with a high yield by the expression and purification methods as involved in the invention.
  • the term “identity” refers to the match degree between two polypeptides or between two nucleic acids.
  • two sequences for comparison have the same base or amino acid monomer sub-unit at a certain site (e.g., each of two DNA molecules has an adenine at a certain site, or each of two polypeptides has a lysine at a certain site)
  • the two molecules are identical at the site.
  • the percent identity between two sequences is a function of the number of identical sites shared by the two sequences over the total number of sites for comparison ⁇ 100. For example, if 6 of 10 sites of two sequences are matched, these two sequences have an identity of 60%.
  • DNA sequences CTGACT and CAGGTT share an identity of 50% (3 of 6 sites are matched).
  • the comparison of two sequences is conducted in a manner to produce maximum identity.
  • Such alignment can be conducted by using a computer program such as Align program (DNAstar, Inc.) which is based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453, 1970).
  • the percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • conservative substitution refers to amino acid substitutions which would not negatively affect or change the biological activity of a protein/polypeptide comprising the amino acid sequence.
  • a conservative substitution may be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions wherein an amino acid residue is substituted with another amino acid residue having a similar side chain, for example, a residue similar to the corresponding amino acid residue physically or functionally (such as, having similar size, shape, charges, chemical properties including the capability of forming covalent bond or hydrogen bond, etc.).
  • the families of amino acid residues having similar side chains have been defined in the art.
  • amino acids having alkaline side chains for example, lysine, arginine and histidine
  • amino acids having acidic side chains for example, aspartic acid and glutamic acid
  • amino acids having uncharged polar side chains for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids having nonpolar side chains for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • amino acids having ⁇ -branched side chains such as threonine, valine, isoleucine
  • amino acids having aromatic side chains for example, tyrosine, phenylalanine, tryptophan, histidine.
  • a corresponding amino acid residue is preferably substituted with another amino acid residue from the same side-chain family.
  • Methods for identifying amino acid conservative substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).
  • E. coli expression system refers to an expression system consisting of E. coli (strain) and a vector, wherein the E. coli (strain) includes, but are not limited to: GI698, ER2566, BL21 (DE3), B834 (DE3), BLR (DE3), etc., which are available on the market.
  • the term “vector” refers to a nucleic acid vehicle which can have a polynucleotide inserted therein.
  • the vector allows for the expression of the protein encoded by the polynucleotide inserted therein, the vector is called an expression vector.
  • the vector can have the carried genetic material elements expressed in a host cell by transformation, transduction, and transfection into the host cell.
  • Vectors are well known by a person skilled in the art, including, but not limited to plasmids, phages, cosmids and the like.
  • a truncated HPV52 L1 protein refers to the protein with one or more amino acids deleted at the N- and/or C-terminal of wild-type HPV52 L1 protein, wherein the example of the wild-type HPV52 L1 protein includes, but is not limited to, the full-length L1 proteins such as ACX32362.1, Q05138.2 or ABU55790.1 in NCBI database.
  • the amino acid sequence of wild-type HPV52 L1 protein may be as set forth in SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29.
  • a gene fragment of a truncated HPV52 L1 protein refers to the gene fragments with the nucleotide(s) encoding one or more amino acids deleted at 5′ or 3′ terminal of the wild-type HPV52 L1 gene, wherein the full-length gene sequence of the wild-type HPV52 L1 gene includes, but is not limited to, the following sequences: EU077195.1, EU077194.1, FJ615303.1 in NCBI database.
  • the term “pharmaceutically acceptable carriers and/or excipients” refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with subjects and active ingredients, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro A R, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to pH adjusting agents, surfactants, adjuvants, and ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to: anion surfactants, cation surfactants, or non-ionic surfactants (for example, Tween-80); adjuvants include, but are not limited to, aluminum adjuvants (for example, aluminum hydroxide) and Freund's adjuvants (for example, Freund's complete adjuvant); and ionic strength enhancers include, but are not limited to, NaCl.
  • surfactants include, but are not limited to: anion surfactants, cation surfactants, or non-ionic surfactants (for example, Tween-80)
  • adjuvants include, but are not limited to, aluminum adjuvants (for example, aluminum hydroxide) and Freund's adjuvants (for example, Freund's complete adjuvant)
  • ionic strength enhancers include, but are not limited to, NaCl.
  • an effective amount refers to an amount that can effectively achieve the intended purpose.
  • an amount effective for preventing a disease refers to an amount effective for preventing, suppressing, or delaying the occurrence of a disease (such as HPV infection). The determination of such an effective amount is within the ability of a person skilled in the art.
  • chromatography includes, but is not limited to: ion exchange chromatography (e.g. cation-exchange chromatography), hydrophobic interaction chromatography, absorbent chromatography (e.g. hydroxyapatite chromatography), gel filtration chromatography (gel exclusion chromatography), and affinity chromatography.
  • the truncated HPV52 L1 proteins according to the invention may be obtained preferably by the following steps:
  • the buffers used in the methods of the invention are well known in the art, including, but not limited to Tris buffers, phosphate buffers, HEPES buffers, MOPS buffers, etc.
  • the disrupting of the host cell can be accomplished by methods well known by a person skilled in the art, including, but not limited to homogenizer disrupting, ultrasonic treatment, grinding, high pressure extrusion, lysozyme treatment, etc.
  • the salts used in the methods of the invention include, but are not limited to: one or more of acidic salts, basic salts, neutral salts, for example, alkali metal salts, alkaline-earth metal salts, ammonium salts, hydrochlorides, sulfates, bicarbonates, phosphate salts or biphosphates, especially NaCl, KCl, NH 4 Cl, (NH 4 ) 2 SO 4 . NaCl is particularly preferred.
  • the reductant used in the methods of the invention includes, but is not limited to, DTT and 2-mercaptoethanol, at an amount including, but not limited to, 10-100 mM.
  • the HPV52 VLPs according to the invention may be produced by the following steps: further purifying the truncated HPV52 L1 protein with a purity of at least 50% as described above by e.g. a chromatography, and thereby obtaining a purified truncated protein solution; and removing the reductant from the solution to obtain the HPV52 VLPs.
  • Methods for removing the reductant are known in the art, including, but not limited to, dialysis, ultrafiltration, and chromatography.
  • the expression systems useful for preparing HPV VLPs include eukaryotic and prokaryotic expression systems.
  • HPV L1 proteins expressed in eukaryotic expression systems show little conformational difference from that of the native virus, and can self-assemble into VLPs. In most cases, VLPs with a correct conformation can be obtained by simple purification. Nevertheless, eukaryotic expression systems, such as the baculovirus and yeast expression systems, are difficult to be applied to large-scale industrial production due to shortcomings such as low expression levels and high culturing costs.
  • Prokaryotic expression systems such as E. coli systems
  • HPV L1 proteins when expressed in E. coli system, HPV L1 proteins usually lose their native conformations and are expressed in a form of inclusion bodies in the precipitant.
  • renaturation of the protein from inclusion bodies is still a challenge worldwide. Due to the difficulty and inefficiency of renaturation, this method is limited to small-scale lab research and cannot be applied to the large-scale obtainment of VLPs with a correct conformation from the inclusive bodies.
  • HPV L1 protein may be expressed in a soluble form with a correct conformation in E. coli , their expression levels are low.
  • E. coli expression systems are used in the invention to express the N-truncated HPV52 L1 protein, which ensures a high expression level.
  • the truncated protein is selectively precipitated from the E. coli lysate supernatant under mild conditions. The truncated protein is then redissolved in a salt buffer to significantly improve its purity while still retaining its correct conformation.
  • the truncated protein solution thus obtained can be further purified directly by chromatography such as ion-exchange and hydrophobic exchange chromatography so as to obtain the protein of interest with a high purity (such as a purity up to 80%). Further, the purified, truncated protein obtained from these steps, can self-assemble into VLP with good immunogenicity and the ability to induce neutralization antibodies of a high titer against HPV52, which is a good vaccine for preventing HPV52 infection in human.
  • the truncated protein of the invention can be expressed in E. coli expression systems on a large scale whilst retaining the antigenicity, immunogenicity, and particle self-assembly ability of the full-length HPV52 L1 protein. Expensive enzymes are not required in the preparation methods used in the invention, i.e. the cost is low. Furthermore, since the truncated protein is not subjected to the intensive procedures of denaturation and renaturation during purification, the loss of the protein is low and the yield is high.
  • the VLPs formed from the truncated protein can induce the generation of protective antibodies against HPV at a high titer and can be applied to the preparation of vaccines. Thus, the truncated protein of the invention and the preparation method thereof can be applied to large-scale industrial production, and makes the large-scale industrial production of vaccines for cervical cancer possible.
  • FIG. 1 shows the SDS-PAGE result of the HPV52N40C-L1 protein obtained during different steps of Example 3 of the invention.
  • Lane M protein molecular weight marker
  • Lane 1 supernatant of disrupted bacteria (i.e. the supernatant obtained by centrifuging the disrupted bacteria)
  • Lane 2 precipitate product free of salts (i.e. the precipitate obtained by centrifugation after dialysis)
  • Lane 3 re-dissolved supernatant (i.e. the supernatant obtained by centrifuging the solution resulted from re-dissolving the precipitate product free of salts)
  • Lane 4 precipitant obtained after re-dissolution (i.e.
  • FIG. 2 shows the SDS-PAGE result of HPV52N40C-L1 purified by cation exchange chromatography and CHT-II in Example 4.
  • Lane M protein molecular weight marker
  • Lane 1 HPV52N40C-L1 purified by the method of Example 4 (the loading volume was 10 ⁇ L)
  • Lane 2 HPV52N40C-L1 purified by the method of Example 4 (the loading volume was 20 ⁇ L).
  • the result showed that HPV52N40C-L1 protein purified by the cation exchange chromatography and CHT-II of Example 4 reached a purity of about 98%.
  • TEM transmission electron microscopy
  • FIG. 4 shows cryo-electron microscopy photograph of HPV52N40C-L1 VLPs obtained in Example 5 and its reconstructed three-dimensional structure, as described in Example 6.
  • FIG. 4A HPV52N40C-L1 VLPs
  • FIG. 4B the reconstructed three-dimensional structure of HPV52N40C-L1 VLPs.
  • HPV52N40C-L1 VLP Unlike general icosahedral capsids meeting quasi-equivalent principle, all the subunits in the structure of HPV52N40C-L1 VLP were pentamers, no hexamers were found, and the VLP had a most peripheral diameter of 60 nm.
  • the structure was similar to the three-dimensional structures of the previously reported native HPV viral particles and the HPV VLPs from eukaryotic expression systems (such as, poxvirus expression system) (Baker T S, Newcomb W W, Olson N H. et al. Biophys J. (1991), 60(6): 1445-1456; Hagensee M E, Olson N H, Baker T S, et al. J Virol. (1994), 68(7): 4503-4505; Buck C B, Cheng N, Thompson C D. et al. J Virol. (2008), 82(11):5190-7).
  • FIG. 5 shows the dynamic light-scattering measurement result of HPV52N40C-L1 VLPs obtained in Example 5, as described in Example 6. The result showed that HPV52N40C-L1 VLPs had a hydrodynamic radius of 24.39 nm and a particle assembly rate of 100%.
  • FIG. 6 shows neutralization titers of antibodies in serum at different stages after vaccination of rabbits with HPV52N40C-L1 VLPs as determined in Example 7. Vaccination times are indicated with arrows. The neutralization titers of antibodies increased significantly one month after the first vaccination, and reached a peak level of 10 5 after a booster.
  • FIG. 7 shows the SDS-PAGE results of the HPV52 L1 proteins having 27, 35, 38 or 42 amino acids truncated at the N-terminal, respectively, i.e. HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, HPV52N42C-L1 (their amino acid sequences were set forth in SEQ ID NOs: 1, 7, 10 and 13, respectively), as obtained in Example 8.
  • Lane M protein molecular weight marker
  • Lane 1 HPV52N27C-L1 protein (the loading volume was 10 ⁇ L)
  • Lane 2 HPV52N35C-L1 protein (the loading volume was 10 ⁇ L)
  • Lane 3 HPV52N38C-L1 protein (the loading volume was 10 ⁇ L)
  • Lane 4 HPV52N42C-L1 protein (the loading volume was 10 ⁇ L).
  • the results showed that the truncated proteins, i.e. HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, HPV52N42C-L1, as obtained in Example 8, reached a purity of about 98%.
  • FIG. 8A HPV52N27C-L1 VLPs
  • FIG. 8B HPV52N35C-L1 VLPs
  • FIG. 8C HPV52N38C-L1 VLPs
  • FIG. 8D HPV52N42C-L1 VLPs.
  • the results showed that a large number of VLPs with a radius of about 25 nm were observed in visual field in the four figures, wherein the particle size was consistent with the theoretic size and the particles were homogenous.
  • FIG. 9 shows the dynamic light-scattering measurement results of HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, and HPV52N42C-L1 VLPs obtained in Example 8.
  • FIG. 9A HPV52N27C-L1 VLPs
  • FIG. 9B HPV52N35C-L1 VLPs
  • FIG. 9C HPV52N38C-L1 VLPs
  • FIG. 9D HPV52N42C-L1 VLPs.
  • HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, and HPV52N42C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of 100%.
  • Sequence 1 (SEQ ID NO: 1): 1 MSVWRPSEAT VYLPPVPVSK VVSTDEYVSR TSIYYYAGSS RLLTVGHPYF SIKNTSSGNG 61 KKVLVPKVSG LQYRVFRIKL PDPNKFGFPD TSFYNPETQR LVWACTGLEI GRGQPLGVGI 121 SGHPLLNKFD DTETSNKYAG KPGIDNRECL SMDYKQTQLC ILGCKPPIGE HWGKGTPCNN 181 NSGNPGDCPP LQLINSVIQD GDMVDTGFGC MDFNTLQASK SDVPIDICSS VCKYPDYLQM 241 ASEPYGDSLF FFLRREQMFV RHFFNRAGTL GDPVPGDLYI QGSNSGNTAT VQSSAFFPTP 301 SGSMVTSESQ LFNKPYWLQR AQGHNNGICW GNQLFVTVVD TTRSTNMTLC AEVKKESTYK 361
  • the molecular biological experimental methods and immunological assays used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3 rd Edition, John Wiley & Sons, Inc., 1995, or in accordance with the product instructions.
  • the reagents and instruments used in the present invention without marking out their manufacturers are all conventional products commercially available from markets. Those skilled in the art understand that the examples are used for illustrating the present invention, but not intended to limit the protection scope of the present invention.
  • the full-length HPV52 L1 Gene (SEQ ID NO: 30) as a template was synthesized by Shanghai Boya Bio Co.
  • the synthesized gene fragment has a full length of 1590 bp.
  • the polynucleotides encoding the truncated HPV52 L1 proteins according to the invention were prepared.
  • the synthesized full-length HPV52 L1 gene was used as the template for the PCR reaction.
  • the forward primer was 52N40F: 5′-CAT ATg CCC GTG CCC GTG AGC AAG-3′ (SEQ ID NO: 31), at the 5′ terminal of which the restriction endonuclease NdeI site CAT ATG was introduced, wherein ATG was the initiation codon in E. coli system.
  • the reverse primer was 52CR: 5′-GTC GAC TCA CCT CTT CAC CTT CTT C-3′ (SEQ ID NO: 32), at the 5′ terminal of which the restriction endonuclease SalI site was introduced.
  • the PCR reaction was performed in a PCR thermocycler (Biometra T3) under the following conditions:
  • the DNA fragments were obtained after amplification.
  • the PCR products were linked into the commercially available pMD 18-T vector (Takara Biosciences), and were transformed into E. coli . Positive bacterial colonies were screened, and plasmids were extracted. After digestion with NdeI/SalI, it was identified that positive clones, designated as pMD 18-T-HPV52N40C-L1, were obtained, wherein the truncated HPV52 L1 gene was inserted.
  • the nucleotide sequence of the fragment of interest which was inserted into the plasmid pMD 18-T-HPV52N40C-L1, was determined as SEQ ID NO: 25 by Shanghai Boya Bio Co. using M13 (+)/( ⁇ ) primers, and the amino acid sequence encoded thereby was set forth in SEQ ID NO: 12.
  • the sequence corresponded to a HPV52 L1 protein having 40 amino acids truncated at its N-terminal and no amino acid truncated at its C-terminal, designated as HPV52N40C-L1.
  • the HPV52N40C-L1 gene fragment was obtained by NdeI/SalI digestion of plasmid pMD 18-T-HPV52N40C-L1.
  • the fragment was linked into the prokaryotic expression vector pT0-T7 (purchased from Invitrogen) digested with NdeI/SalI, and was transformed into ER2566 bacteria. Positive bacterial colonies were screened, and plasmids were extracted. After digestion with NdeI/SalI, it was identified that positive clones, designated as pT0-T7-HPV52N40C-L1, were obtained, wherein the fragment of interest was inserted.
  • plasmid pT0-T7-HPV52N40C-L1 (0.15 mg/ml) was used to transform 40 ⁇ L competent E. coli ER2566 (purchased from Invitrogen) prepared by the Calcium chloride method, and then the bacteria were plated on solid LB medium (the components of the LB medium: 10 g/L peptone, 5 g/L yeast powder, and 10 g/L NaCl, the same as below) containing kanamycin (at a final concentration of 100 mg/ml, the same as below). The plates were statically incubated at 37° C. for about 10-12 h until single colonies could be observed clearly.
  • the E. coli solution carrying the recombinant plasmid pTO-T7-HPV52N40C-L1 at ⁇ 70° C. as prepared in Example 1 was seeded in 50 mL LB liquid medium containing kanamycin and incubated at 180 rpm and 37° C. for about 12 h. Then, the cultures were transferred to ten flasks (5 ml cultures per flask), each of which contained 500 mL LB medium containing kanamycin, and was incubated in a shaking incubator overnight at 180 rpm and 37° C., as a starter culture.
  • a 50 L fermenter made by Shanghai Baoxing Biological Ltd was used in large-scale culture.
  • PH electrode of the fermenter was calibrated.
  • 30 L LB medium was loaded into the fermenter, in situ sterilized at 121° C. for 30 minutes.
  • Oxygen-dissolved electrode was calibrated, wherein the value was determined as 0 prior to introduction of air after sterilization and as 100% prior to vaccination after introduction of air while stirring at an initial rate of 100 rpm.
  • the culturing temperature was lowered to 25° C. and 4 g IPTG was added to initiate an induction culture of 12 h. Fermentation was halted when the final concentration reached an OD 600 of about 40.
  • the bacteria expressing HPV52N40C-L1 protein were obtained, weighted about 2.5 kg.
  • Bacteria were re-suspended at a proportion of 1 g bacteria corresponding to 10 ml lysis buffer (20 mM Tris buffer pH 7.2, 300 mM NaCl). Bacteria were disrupted by an APV homogenizer (Invensys Group) for five times at a pressure of 600 bar. The homogenate was centrifuged at 13,500 rpm (30,000 g) using JA-14 rotor for 15 min, and the supernatant (i.e. the supernatant of disrupted bacteria) was obtained. The supernatant was subjected to 10% SDS-PAGE. At this stage, the HPV52N40C-L1 protein in the supernatant had a purity of about 10% (see FIG. 1 , Lane 1).
  • the supernatant was dialyzed by a CENTRASETTE 5 Tangential Flow Filter (Pall Co.) running at a pressure of 0.5 psi, a flow rate of 500 ml/min, and a tangential flow rate of 200 mL/min, wherein the membrane retention molecular weight was 30 kDa, the dialysis solution was 10 mM phosphate buffer pH 6.0, and the dialysis volume was three times of the volume of the supernatant.
  • the mixture was centrifuged at 9500 rpm (12,000 g) using JA-10 rotor (Beckman J25 high speed centrifuge) for 20 min, and the precipitate (i.e. the precipitate product free of salts) was collected.
  • the precipitate was re-suspended in 10 mM phosphate buffer (pH 7.0) containing 10 mM DTT and 300 mM NaCl, wherein the volume of the buffer was 1/10 of the volume of the supernatant.
  • the mixture was stirred for 30 min and centrifuged at 13,500 rpm (30,000 g) using JA-14 rotor (Beckman J25 high speed centrifuge) for 20 min.
  • the supernatant and precipitate i.e.
  • the precipitate obtained after re-dissolution were collected.
  • the supernatant passed through a filter membrane with an aperture of 0.22 ⁇ m.
  • the sample obtained i.e. re-dissolved supernatant
  • 30 ⁇ L of 6 ⁇ loading buffer (12% (w/v) SDS, 0.6% (w/v) bromophenol blue, 0.3M Tris-HCl pH 6.8, 60% (v/v) glycerin, 5% (v/v) ⁇ -mercaptoethanol) was added to 150 ⁇ L filtered supernatant, and the resultant solution was mixed homogeneously and was placed in a water bath at 80° C. for 10 min.
  • Buffer 20 mM phosphate buffer pH 8.0, 10 mM DTT
  • Sample 3 L of about 70% pure HPV52N40C-L1 protein solution, as filtered through a filter membrane with an aperture of 0.22 ⁇ m in Example 3.
  • Elution protocol eluting undesired proteins with 500 mM NaCl, eluting the protein of interest with 1000 mM NaCl, collecting eluate eluted with 1000 mM NaCl, and finally getting about 900 mL purified HPV52N40C-L1 sample.
  • Chromatographic media CHT-II (purchased from Bio-Rad)
  • Buffer 20 mM phosphate buffer pH8.0, 10 mM DTT,
  • Sample 1000 mM NaCl elution product obtained in the previous step, diluted to a NaCl concentration of 500 mM with 20 mM phosphate buffer pH 8.0, 10 mM DTT.
  • Elution protocol eluting undesired proteins with 500 mM NaCl, eluting the protein of interest with 1000 mM NaCl, collecting eluate eluted with 1000 mM NaCl, and finally getting 800 mL purified HPV52N40C-L1 sample.
  • Sample Concentration Sample was concentrated to 600 mL by adjusting the tangential flow rate of the tangential flow system to 50 mL/min.
  • Sample buffer was exchanged with 10 L renaturation buffer (20 mM PB (sodium phosphate buffer) pH 6.0, 2 mM CaCl 2 , 2 mM MgCl 2 , 0.5M NaCl, 0.003% Tween-80) thoroughly.
  • the Tangential Flow Filter was run at a pressure of 0.5 psi and a tangential flow rate of 10 mL/min.
  • the renaturation buffer was exchanged with storage buffer (20 mM PB (sodium phosphate buffer) pH 6.5, 0.5M NaCl) with an exchange volume of 20 L.
  • the Tangential Flow Filter was run at a pressure of 0.5 psi and a tangential flow rate of 25 mL/min.
  • the sample was aseptically filtrated with a Pall filter (0.22 ⁇ m), and thereby obtaining HPV52N40C-L1 VLPs.
  • the HPV52N40C-L1 VLPs were stored at 4° C. for further use.
  • HPV52N40C-L1 VLPs obtained in Example 5 were negatively stained with 2% phosphotungstic acid at pH 7.0, and fixed on a copper grid for observation. Results were shown in FIG. 3 . A large number of VLPs with a radius of approximately 25 nm, which were homogenous and in a hollow form, were observed.
  • the three-dimensional structure of HPV52N40C-L1 VLPs was reconstructed by the three-dimensional structure reconstruction experiment using cryo-electron microscopy (Wolf M, Garcea R L, Grigorieff N. et al. Proc Natl Acad Sci USA. (2010), 107(14): 6298-303).
  • cryo-electron microscopy photograph of HPV52N40C-L1 VLPs FIG. 4A
  • 400 homogeneous particles with a diameter of above 50 nm were separately selected for computer refolding and structure reconstruction, thereby obtaining the three-dimensional structure of HPV52N40C-L1 VLPs.
  • the three-dimensional structure obtained was shown in FIG.
  • DynaPro MS/X dynamic light-scattering instrument including a temperature controller (US Protein Solutions Co.) was used for light-scattering measurements.
  • the Regulation algorithm was used in the measurements.
  • the sample was the HPV52N40C-L1 VLPs obtained in Example 5.
  • the sample was passed through a 0.22 ⁇ m filter membrane prior to the measurement.
  • the result was shown in FIG. 5 .
  • the result showed that HPV52N40C-L1 VLPs had a hydrodynamic radius of 24.39 nm.
  • HPV can hardly be cultured in vitro, and the HPV host is strongly specific. Thus, HPV can hardly be propagated in hosts other than human. That is, there was not an appropriate animal model for HPV. Therefore, in order to evaluate the immune protection of HPV vaccines quickly, it is urgent to establish an effective model for in vitro neutralization assays.
  • HPV pseudovirion was formed by expressing HPV L1 and L2 protein in cells, and by packaging episomal viral DNA or reporter plasmids introduced heterologously (Yeager, M. D, Aste-Amezaga, M. et al (2000) Virology (278) 570-7).
  • the concrete methods include methods of recombinant viral expression systems and methods of co-transfection of multi-plasmids. Methods of co-transfection of multi-plasmids were used in the Example exemplarily.
  • HPV systems were made by conventional methods as followed.
  • the calcium phosphate transfection method for 293FT cell line was optimized to obtain a transfection efficiency of up to more than 90%, thereby facilitating large-scale production.
  • the expression plasmid for expressing HPV structural proteins was codon-optimized to express HPV L1 and L2 gene efficiently in mammalian cells, thereby facilitating high efficient assembly of pseudovirion.
  • Plasmid p52L1h (the pAAV vector carrying the nucleotide sequence encoding HPV52 L1 protein (NCBI database, Accession Number: Q05138)
  • plasmid p52L2h (the pAAV vector carrying the nucleotide sequence encoding HPV52 L2 protein (NCBI database, Accession Number: P36763)
  • plasmid pN31-EGFP carrying green fluorescent protein gene were purified by CsCl density gradient centrifugation, wherein said pN31-EGFP and said pAAV vectors were donated by Professor John T. Schiller of NIH. Methods for purifying plasmids using CsCl density gradient centrifugation were well known in the art (see The Molecular Cloning Experiment Guide, 3rd edition).
  • 293FT cells (Invitrogen) cultured on a 10 cm cell culture plate were co-transfected with the purified p52L1h, p52L2h and pN31-EGFP (40 ⁇ g for each) by calcium phosphate transfection method.
  • Calcium phosphate transfection method was well known in the art (see The Molecular Cloning Experiment Guide, 3rd edition).
  • p52L1h, p52L2h and pN31-EGFP (40 ⁇ g for each) were added to the mixture of 1 mL HEPES solution (125 ⁇ L 1M HEPES pH7.3 per 50 mL deionized water, stored at 4° C.) and 1 mL 0.5M CaCl 2 solution.
  • the positive region was defined as the cell region having a GFP signal determined by flow cytometry at least 10 times higher than the signal of the control cells.
  • Neutralization titer of antibodies was defined as the highest dilution fold under which the infection-inhibition percentage reached above 50%. Antibodies were considered as having neutralizing capacity if their infection-inhibition percentage was above 50% after 50 times dilutions.
  • Rabbits were used to evaluate the immune protection of the HPV52 VLPs according to the invention.
  • Animals for vaccination were female rabbits (general grade), 6-8 weeks old, purchased from the Disease Prevention and Control Center of Guangxi province.
  • HPV52N40C-L1 VLPs (at a concentration of 0.1 mg/ml) prepared in Example 5, were mixed with equal volume of complete Freund's Adjuvant for the first vaccination, or with equal volume of incomplete Freund's Adjuvant for the booster.
  • the vaccination procedure was as followed: the first vaccination at Week 0, and the booster at Weeks 4 and 10, respectively.
  • Rabbits were vaccinated via muscle injection, with 100 ⁇ g per rabbit for the first vaccination, and with 50 ⁇ g per rabbit for the booster.
  • peripheral venous blood was collected every week, and serum was separated and stored for test.
  • the neutralization titers of antibodies against HPV52 pseudovirion in the rabbit serum were determined by the method above.
  • FIG. 6 showed that neutralization titers of antibodies in serum at different stages after vaccination of rabbits with HPV52N40C-L1 VLPs. Vaccination times were indicated by arrows. It could be seen that the neutralization titers of antibodies increased significantly one month after the first vaccination, and reached a peak level of 10 5 after one booster. It showed that HPV52N40C-L1 VLPs obtained by the methods as described in Examples 1-5 had good immunogenicity, could induce the generalization of neutralization antibodies against HPV52 with a high titer in animals, and could be used as an effective vaccine for the prevention of HPV52 infection. In addition to Freund's Adjuvant, other adjuvants well known in the art might also be used in the vaccines, for example, aluminum hydroxide or aluminum phosphate adjuvants.
  • HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, and HPV52N42C-L1 having 27, 35, 38 or 42 amino acids truncated at the N-terminal were prepared and purified basically by the methods as described in Examples 1-4.
  • the four proteins thus obtained had a purity of above 98% (see FIG. 7 ).
  • HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1 and HPV52N42C-L1 proteins were assembled into VLPs basically by the method as described in Example 5, respectively, designated as HPV52N27C-L1 VLPs, HPV52N35C-L1 VLPs, HPV52N38C-L1 VLPs, and HPV52N42C-L1 VLPs, respectively.
  • HPV52N27C-L1 VLPs, HPV52N35C-L1 VLPs, HPV52N38C-L1 VLPs, and HPV52N42C-L1 VLPs were subjected to transmission electron microscopy and dynamic light scattering observation basically by the method as described in Example 6, respectively.
  • the results were shown in FIG. 8 and FIG. 9 .
  • FIG. 8 showed that the truncated proteins could form a large number of VLPs with a radius of about 25 nm, wherein the particle size was consistent with the theoretic size and the particles were homogenous.
  • FIG. 9 showed that HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, HPV52N42C-L1 VLPs had a hydrodynamic radius of about 25 nm and a particle assembly rate of 100%.
  • HPV52N27C-L1, HPV52N35C-L1, HPV52N38C-L1, HPV52N42C-L1 VLPs obtained in the invention also had good immunogenicity, could induce the generalization of neutralization antibodies with a high titer in animals, and therefore could be used as an effective vaccine for the prevention of HPV infection.

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CN105985934A (zh) * 2015-08-18 2016-10-05 北京康乐卫士生物技术股份有限公司 一种二价、三价或多价hpv假病毒及其应用
WO2017092710A1 (fr) * 2015-12-04 2017-06-08 厦门大学 Mutant de la protéine l1 du virus du papillome humain de type 58
CN106831959B (zh) * 2015-12-04 2019-11-05 厦门大学 一种人乳头瘤病毒33型l1蛋白的突变体
CN107557347B (zh) * 2016-06-30 2022-03-29 中国科学院上海巴斯德研究所 肠道病毒71型的新型病毒样颗粒、其制备方法及应用
WO2021013071A1 (fr) * 2019-07-19 2021-01-28 神州细胞工程有限公司 Composition polyvalente induisant l'immunogénicité pour papillomavirus humain
WO2021013078A1 (fr) * 2019-07-19 2021-01-28 神州细胞工程有限公司 Protéine l1 de papillomavirus humain de type l1 chimérique
CN114539365B (zh) * 2020-11-26 2023-12-01 中国医学科学院基础医学研究所 一种改造的人乳头瘤病毒52型l1蛋白及其用途

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000054730A2 (fr) 1999-03-18 2000-09-21 The President & Fellows Of Harvard College Compositions de vaccins contre le papillomavirus humain et procedes associes
CN1478790A (zh) 2002-08-30 2004-03-03 马润林 乳头瘤病毒衣壳蛋白的原核制备和应用
CN1934131A (zh) 2004-03-24 2007-03-21 默克公司 Hpv52 l1在酵母中的优化表达
CN101153280A (zh) 2006-09-29 2008-04-02 厦门大学 从原核生物中纯化人乳头瘤病毒晚期蛋白l1的方法
CN101293918A (zh) 2007-04-29 2008-10-29 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒16型l1蛋白
CN101343314A (zh) 2007-05-29 2009-01-14 厦门大学 截短的人乳头瘤病毒11型l1蛋白
CN101343315A (zh) 2007-05-29 2009-01-14 厦门大学 截短的人乳头瘤病毒6型l1蛋白
CN101570571A (zh) 2007-04-29 2009-11-04 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒18型l1蛋白

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000054730A2 (fr) 1999-03-18 2000-09-21 The President & Fellows Of Harvard College Compositions de vaccins contre le papillomavirus humain et procedes associes
US6551597B1 (en) 1999-03-18 2003-04-22 President & Fellows Of Harvard College Vaccine compositions for human papillomavirus
CN1478790A (zh) 2002-08-30 2004-03-03 马润林 乳头瘤病毒衣壳蛋白的原核制备和应用
CN1934131A (zh) 2004-03-24 2007-03-21 默克公司 Hpv52 l1在酵母中的优化表达
US7744892B2 (en) * 2004-03-24 2010-06-29 Merck Sharp & Dohme Corp. Optimized expression of HPV 52 L1 in yeast
CN101153280A (zh) 2006-09-29 2008-04-02 厦门大学 从原核生物中纯化人乳头瘤病毒晚期蛋白l1的方法
CN101293918A (zh) 2007-04-29 2008-10-29 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒16型l1蛋白
CN101570571A (zh) 2007-04-29 2009-11-04 北京万泰生物药业股份有限公司 截短的人乳头瘤病毒18型l1蛋白
EP2147926A1 (fr) 2007-04-29 2010-01-27 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Protéines 18 l1 de type papillomavirus humain tronque
EP2154147A1 (fr) 2007-04-29 2010-02-17 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Protéine l1 tronquée du papillomavirus 16 humain
CN101343314A (zh) 2007-05-29 2009-01-14 厦门大学 截短的人乳头瘤病毒11型l1蛋白
CN101343315A (zh) 2007-05-29 2009-01-14 厦门大学 截短的人乳头瘤病毒6型l1蛋白

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
Baker et al.; "Structures of bovine and human papillomaviruses"; Analysis by cryoelectron microscopy and three-dimensional image recontruction; Biophys. J.; vol. 60; Dec. 1994; pp. 1445-1456.
Banks et al.; "Expression of Human Papillomavirus Type 6 and Type 16 Capsid Proteins in Bacteria and Their Antigenice Characterization", J. gen. Vivol., 1987; 68; pp. 3081-3089.
Brummell et al.; "Probing the Combining Site of an Anti-Carbohydrate Antibody by Saturation-Mutagenesis: Role of the Heavy-Chain CDR3 Residues"; Biochemistry 1993; 32; pp. 1180-1187.
Buck et al.; "Arrangement of L2 within the Papillomavirus Capsid"; Journal of Virology, Jun. 2008; vol. 82; No. 11; pp. 5190-5197.
Burks et al.; "In Vitro Scanning saturation mutagenesis of an antibody binding pocket"; Proc. Natl. Acad. Sci, USA, Jan. 1997; vol. 94, pp. 412-417.
Chen et al. J. Mol. Biol. (2001) 307, 173-182. *
Chen et al.; "Papillomavirus capsid protein expression in Escherichia coli purification and assembly of HPV11 and HPV16 L1", Journal of Molecular Biology, Academic Press, United Kingdom, vol. 307, No. 1, Mar. 16, 2001, pp. 173-182.
European Search Reported dated Nov. 15, 2013 for Appln No. 11800199.9.
Fey et al. Demonstration of in vitro synthesis of human papilloma viral proteins from hand and foot warts. J Invest Dermatol. Jun. 1989;92(6):817-24. *
GenBank: ACV84004.1. major capsid protein L1 [Human papillomavirus type 16]. http://www.ncbi.nlm.nih.gov/protein/ACV84004.1. Dated Sep. 22, 2009. *
GenBank: ADR90828.1, Sequence 2 from U.S. Pat. No. 7,744,892, http://www.ncbi.nlm.nih.gov/protein/ADR90828.1, dated Dec. 12, 2010. *
GenBank: AEI61597.1. late protein L1 [Human papillomavirus type 52]. Dated Jun. 27, 2011. http://www.ncbi.nlm.nih.gov/protein/AEI61597.1. *
GenBank: CAA52590.1. late protein [Human papillomavirus type 52]. http://www.ncbi.nlm.nih.gov/protein/397045. Apr. 18, 2005. *
Hagensee et al.; "Three-Dimensional Structure of Vaccinia Virus-Produced Human Papillomavirus Type 1 Capsids"; Journal of Virology, Jul. 1994; vol. 68; No. 7; pp. 4503-4505.
Huhti et al.A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles. Arch Virol (2010) 155:1855-1858. *
International Search Report for PCT/CN2011/076763.
Kelsall et al.; "Expression of the major capsid protein of human papillomavirus type 16 in Escherichia coli", Journal of Virological Methods, 53; 1995; pp. 75-90.
Kirnbauer et al.; "Papillomavirus L1 major capsid protein self-assembles into virus-like particesl that are highly immunogenic"; Pro. Natl. Acad. Sci. USA; vol. 89; Dec. 1992; pp. 12180-12184.
Kobayashi et al.; "Tryptophan H33 plays an important role in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody"; Protein Engineering; 1994; vol. 12; No. 10; pp. 879-884.
Kozak. Point Mutations Define a Sequence Flanking the AUG Initiator Codon That Modulates Translation by Eukaryotic Ribosomes. Cell, vol. 44, 283-292, Jan. 31, 1986. *
Li et al.; " Expressions of Human Papillomavirus Type 11 L1 Capsid Protein in Escherichia coli: Characterizaiton of Protein Domains Involved in DNA Binding and Capsid Assembly"; Journal of Virology, Apr. 1997;vol. 71; No. 4; pp. 2988-2995.
Ma et al. Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion. Protein Expr Purif. Nov. 2007;56(1):72-9. Epub May 29, 2007. *
Ma et al.; "Increasing the expression levels of papillomavirus major capsid protein in Escherichia coli by N-terminal deletion", Protein Expression and Purification, Academic Press, Sand Diego, CA, vol. 56, No. 1, Oct. 3, 2007, pp. 72-79.
Machine English translation of CN 101153280 A. *
Myers et al.; "Optimal alignments in linear space"; Comput Appl Bioscience; Mar. 1988;4(1):11-7; pp. 1-13.
Needleman et al.; "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins"; J. Mol. Bio, 1970; 48; pp. 443-453.
Wolf et al.; "Subunit interactions in bovine papillomavirus"; Apr. 6, 2010; vol. 107; No. 14; pp. 6298-6303.
Zemskovaa et al. Transient expression of deletion mutants of the herpes simplex virus thymidine kinase-encoding gene in mouse fibroblast cells. Gene. vol. 106, Issue 2, Oct. 15, 1991, pp. 249-253. *

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